Endotoxin (lipopolysaccharide; LPS) is the major pathogenic factor of gram-negative septic shock. Endotoxin-induced shock and death may be associated with host overproduction of tumor necrosis factor alpha (TNF-(). In the search for new anti-endotoxin molecules, we studied the endotoxin-neutralizing capacity of a human lactoferrin-derived 33-mer synthetic peptide (GRRRRSVQWCAVSQPEATKCF-QWQRNMRKVRGP, designated LF-33) representing the minimal sequence for the lactoferrin binding to glycosaminoglycans. LF-33 inhibited the coagulation of the Limulus amebocyte lysate and the secretion of TNF-( by RAW264.7 cells, a mouse macrophage cell line, induced by lipid A and four different LPS. Its inhibitory capability was comparable to that of polymyxin B. The first 6 residues (GRRRRS) at the N-terminus of LF-33 were critical for its anti-LPS activity. The endotoxin-neutralizing capacity of LF-33 and polymyxin B were attenuated in presence of human serum. Co-injection of E. coli LPS (125 ng) with LF-33 (2.5 ug) dramatically reduced the lethality of LPS in the galactosamine-sensitized mouse model. Significant protection of the mice against lethal LPS challenge was also observed when LF-33 (100 ug) was given intravenously after intra-peritoneal injection of LPS. The protection was correlated with reduction of the mouse serum TNF-( levels. These results thus demonstrate the endotoxin-neutralizing capability of LF-33 in vitro and in vivo and its potential use for treatment of endotoxin-induced septic shock. Endotoxin is the major pathogenic factor of gram-negative sepsis and septic shock. Identifying anti-endotoxin agents is not only important for treating this life threatening illness, but may also be useful for reducing the side effects of bacterial vaccines containing unavoidable endotoxin impurities.